Sulphate-reducing bacteria-mediated pyrite formation in the Dachang Tongkeng tin polymetallic deposit, Guangxi, China

Mediation by sulphate-reducing bacteria (SRB) is responsible for pyrite (FeS2) formation. The origin of the Dachang tin polymetallic ore field is related to the mineralisation of submarine hydrothermal vent sediments. Here, we investigated SRB in these ores via morphological, chemical, and isotopic analyses. Polarised and scanning electron microscopy indicated that trace SRB fossils in the metal sulphide ore were present in the form of tubular, beaded, and coccoidal bodies comprising FeS2 and were enclosed within a pyrrhotite (FeS) matrix in the vicinity of micro-hydrothermal vents. The carbon (C), nitrogen (N), and oxygen (O) contents in the FeS2 synthesised by SRB were high, and a clear biological Raman signal was detected. No such signals were discerned in the peripheral FeS. This co-occurrence of FeS, FeS2, and the remains of bacteria (probably chemoautotrophic bacteria) was interpreted as the coprecipitation process of SRB-mediated FeS2 formation, which has, to the best of our knowledge, not been reported before. Our study also illustrates that combined energy-dispersive X-ray spectroscopy, Raman spectroscopy, and isotopic analysis can be used as a novel methodology to document microbial-mediated processes of mineral deposition in submarine hydrothermal vent ecology on geological time scales.


Geological setting
The Dachang tin polymetallic field is a world-renowned non-ferrous metal production area, with large proven resources of tin (Sn; 1.47 million tonnes), Zn (6.80 million tonnes), Pb (1.76 million tonnes), Sb (1.38 million tonnes), and Cu (0.37 million tonnes), as well as associated economically viable and rare elements such as indium (In), cadmium (Cd), and gallium (Ga) 9,10 . The ore-bearing strata of the deposit are Devonian limestone, reefal limestone, siliceous rock, and shale; exposed Late Cretaceous granite porphyry and diorite porphyrite veins exist in the mining area as well. It has been suggested that the deposit is of hydrothermal or composite origin, derived from Late Cretaceous granite [34][35][36] ; however, other researchers reported that the formation of the deposit was related to the mineralisation of submarine vent sediments 31-33 . Regional geology. The Youjiang Basin, located on the southwest edge of the Yangtze Craton in southwest China, was formed by composite parts of several tectonic units, including the Yangtze, North Vietnam, and Simao blocks. The Dachang Sn polymetallic ore field in Guangxi is located in the far northeast of the Youjiang Basin (Fig. 1a). The Youjiang Basin evolved in two stages: the Hercynian (Devonian-Permian) saw a passive continental margin rift stage, and the Indosinian (Early-Middle Triassic) saw a back-arc rift basin stage. In the Devonian, a NW-SE faulted sub-basin was formed inside the Youjiang Basin, in which the southwest margin of the Yangtze Craton underwent extensional faulting 37 . In the early Middle Devonian (Nabiao Formation period), reefs developed in the Dachang area with local underwater uplifts. Synsedimentary faults developed in the Dachang area from the Late Devonian Liujiang to Wuzhishan periods, and siliceous rocks and banded limestone were widely deposited 31 . Some studies have shown that there were two stages of submarine hydrothermal vent sedimentation and mineralisation in the Late Devonian Dachang Sn polymetallic ore field. In the first stage, the Yanjiang Formation strata (D3l; Fig. 1b), dominated by banded siliceous rocks, were formed, and the No. 92 cassiterite-sulphide type orebody was produced. In the second stage, the Wuzhishan Formation strata (D3w; Fig. 1b), primarily composed of banded marble and siliceous rocks, was formed, and the No. 91 cassiteritesulphide type orebody was produced 31,38,39 . Ore deposit geology. The ore-bearing strata are dominated by sandstone, shale, and carbonate rocks, with local carbonaceous mudstone and siliceous rocks. The ore field is primarily composed of five Sn polymetallic deposits, namely Tongkeng (Sn-Zn-Pb), Gaofeng (Sn-Zn-Pb), Dafulou (Sn-Zn), Kangma (Sn-Zn), and Huile (Sn-Zn). It is a non-ferrous metal ore field with one of the largest Sn polymetallic reserves in the world 9 . The Tongkeng Sn polymetallic deposit is located in the western part of the Dachang ore field and has the largest non-ferrous metal reserves in this field. The central part of the Tongkeng deposit is interspersed with granitic porphyry dikes (eastern dikes). Zircon U-Pb dating of the eastern dikes gives an age of 91 ± 1 Ma (i.e., Late Cretaceous 40 ). The No. 92, No. 91, veinlet zone, and large vein zone orebodies are located to the west of the eastern dikes, whereas the No. 96, No. 95, and No. 94 orebodies are located to the east of the eastern dikes (Fig. 1b).
The No. 92 orebody is characterised by laminar-banded, network-vein, nodular, and a small amount of interlayer vein mineralisation. The main ore minerals are sphalerite and FeS 2 ; secondary ore minerals are cassiterite, arsenopyrite, and FeS; gangue minerals are primarily quartz, followed by calcite (CaCO 3 ), tourmaline, and plagioclase.
The No. 91 orebody is primarily composed of laminar-banded and NE-trending jointed vein-like mineralisation. The main ore minerals are cassiterite, marmatite, arsenopyrite, and FeS, followed by FeS 2 . The main gangue minerals are quartz and tourmaline, followed by CaCO 3 and potassium feldspar.
The orebody of the veinlet zone primarily consists of veinlet mineralisation with local laminar-banded mineralisation. The main ore minerals are marmatite, FeS 2 , and jamesonite, followed by cassiterite, arsenopyrite, FeS, and franckeite. The main gangue minerals are CaCO 3 , quartz, and tourmaline.
The orebody in the large vein zone is mineralised primarily by joint veins. The main ore minerals are marmatite, FeS 2 , jamesonite, and franckeite, followed by cassiterite and arsenopyrite.

Results
Mineralogical and geochemical signatures of hydrothermal vent sediments. Polarised microscopy and scanning electron microscopy of well-polished surfaces of the hydrothermal vent sediments showed that FeS 2 formed tubular ( Fig. 2a- www.nature.com/scientificreports/ remnants ( Fig. 2c-d). This multi-stage mineralisation indicates that FeS was formed in the earliest metallogenic process and that the formation environment of FeS 2 was related to the Late Devonian mineralisation of submarine hydrothermal vent sediments. Numerous pores were filled with carbonate minerals in both samples ( Fig. 2a and c), which may have constituted micro-hydrothermal vents. A similar phenomenon, known as the 'ghosts' of bacterial cells, has been observed in other submarine hydrothermal vent sediments as well as in modern vent settings and is believed to have resulted from bacterial iron accumulation on vestimentiferan tubes [41][42][43] .
Microscopic backscatter imaging and elemental analysis (Fig. 3) were performed in areas typical for the presence of SRB. An evident contrast was present in the mineral contents of FeS, FeS 2 , and non-metallic minerals in the backscattered electron images (Fig. 3b and c). In the EDX elemental images, areas of FeS showed high Fe and low S values, whereas those of FeS 2 showed low Fe and high S values ( Fig. 3d and e). C, N, and O were less present in FeS areas, all of which instead showed high values in areas of FeS 2 and non-metallic minerals ( Fig. 3f-h).   Fig. 2g and h). Some tubular FeS 2 formations symmetrically develop hemispherical structures on both sides, with radii ranging from 10 to 25 μm, creating a beaded overall morphology (Fig. 2h). Additionally, some FeS 2 existed as individual spherical particles, with diameters varying greatly from 20 to 50 μm (Fig. 2e). This variation in size may be attributed to spatial differences in the three-dimensional morphology of FeS 2 . Coccoidal and beaded FeS 2 often exhibit distinctive layered structures at the edges (Fig. 4b).
In this study, a 70% nitric acid solution was used to etch the potential FeS 2 regions containing SRB to characterise SRB microstructure. Etched interiors uncovered a substantial presence of spherical and filamentous residues, which were predominantly organic in nature due to the strong corrosive properties of concentrated nitric acid ( Fig. 4c-h). Moreover, in the backscattered electron images, these residues exhibited increased contrast in comparison to the FeS 2 particles, further substantiating their association with SRB ( Fig. 4c-h). The embedding of spherical SRB fossils (ranging in size from 250 to 450 nm) within the FeS 2 matrix indicates that these spheres were unadulterated by contaminants (Fig. 4d,e). Similar spherical microorganisms have also been found in modern hydrothermal vent environments from the eastern Manus Basin, where they have been observed at the layered periphery of FeS 2 , which aligns with the locations in which SRB were found in the Roman Ruins black smokers 44 . Additionally, filamentous SRB fossils exhibiting distinct branching patterns were preserved within a small number of tubular FeS 2 structures after acid etching (Fig. 4f). The diameter of individual filaments averaged approximately 120 nm, and a pronounced curvature could be observed at the endpoints of the filamentous fossils, suggesting that they froze in a phase of outward growth (Fig. 4h). There were also instances in which multiple filamentous SRB fossils were preserved together (Fig. 4g).
Raman spectroscopy. Because of being non-destructive, rapid, and convenient, Raman spectroscopy has been extensively used to identify valuable biological remains in sedimentary and metamorphic rocks [45][46][47][48][49] . The method has also been used as an effective tool for determining the microstructure of suspected biological specimens, potential microfossils, or other substances from various geological periods, especially when assessing their carbonaceous composition [50][51][52] .
In this study, Raman spectroscopy analyses were performed on FeS, spheroidal FeS 2 edges, tubular FeS 2 walls, and the inner carbonate minerals of tubular FeS 2 ( Fig. 5; Supplementary information). The locations of  Fig. 3b and c. The laser Raman spectral characteristics of the different types of minerals clearly differed. Peaks of FeS and FeS 2 primarily occurred in the range of 100-700 cm −1 ( Fig. 5a and b), whereas for FeS 2 , several peaks were also observed in the range of 1000-1700 cm −1 , including the D1 and G peaks of carbonaceous material located near 1300 cm −1 and 1590 cm −1 , respectively ( Fig. 5c-g). Among these, the D1 peak is attributed to the presence of incorporated aromatic or benzene clusters, and the G peak is composed of the E2g2 mode of graphite or sp 2 C=C stretching vibrations 53 . No discernible D2 peak was observed in the carbon materials associated with SRB, indicating a limited degree of graphitisation 54 . The absence of a D2 peak is directly influenced by the maximum environmental temperature experienced by the carbon material 51 . Intriguingly, the positions and morphologies of the D and G peaks in the Raman spectra of the carbon materials investigated in this study closely resembled the Raman signals documented in previous studies on kerogen 55,56 . In the case of these kerogens with relatively low maturity, the D2 peak was frequently insignificant or could not distinctively be separated from the G peak ( Fig. 5d and e). This similarity supports the organic origin of the carbon signal detected in the FeS 2 region using EDX analysis. These findings thus further corroborate the identification of the previously observed microstructures as bacterial microfossils 57 .   56 Fe values of FeS 2 isolated from each sample were higher than those of the corresponding FeS. In addition, the δ 13 C -PDB of organic C in the ore showed a strong negative anomaly, with a mean value of − 34.82‰, and the δ 13 C -PDB of CaCO 3 exhibited a weak negative excursion, with a mean value of − 0.40‰ (Table 1).

Discussion
Role of SRB in isotopic fractionation. The reaction between H 2 S generated by the activity of SRB and early-stage FeS to form FeS 2 is a matter of debate 13 . Views on whether S is gained (Fe-S bond is not broken) or Fe is lost (Fe-S bond is broken) in the reaction process have been conflicting 58,59 . However, more recent studies reported that the process involves both loss of Fe and gain of S 60-62 . Our Fe-isotope analyses show that the δ 56 Fe values of FeS 2 isolated from each sample were higher than those of the corresponding FeS (0.45‰ higher on average). We therefore speculate that some 54 Fe isotopes were lost during the SRB-mediated conversion of FeS to FeS 2 , resulting in an increase in the proportion of 56 Fe in the synthesised FeS 2 compared with that of the original FeS. The S-isotope composition of FeS and FeS 2 is related to the environmental S-isotope composition. The supply of H 2 S in hydrothermal vents derives from both inorganic and organic processes [63][64][65] . Thermochemical sulphate reduction (TSR) occurs at high temperatures before the SO 4 2− -containing hydrothermal fluid is ejected from the seabed, resulting in S-isotope fractionation 66,67 . The generated H 2 S gas is enriched in light S isotopes, whereas the ejected hydrothermal SO 4 2− -rich solution is enriched in heavy S isotopes. Bacterial sulphate reduction (BSR) also causes S-isotope fractionation; the S-isotope composition of the resulting H 2 S gas depends on the S-isotope composition of SO 4 2− in the environment and the extent of selective light S-isotope enrichment during cellspecific sulphate reduction 68,69 .
The S-isotope analysis showed that the δ 34 S values of FeS and FeS 2 ranged from − 4.91 to − 4.17‰, with a mean of − 4.58‰ and a standard deviation of 0.28‰ (Table 1). We infer these abnormal negative 34 S values to be related to TSR and BSR. The δ 34 S values of FeS 2 isolated from the samples were higher than those of the corresponding FeS, which is probably related to the utilisation of SO 4 2− with a high 34 S content in SRB-mediated FeS 2 synthesis. Researchers have analysed the S-isotope composition of barite (BaSO 4 ) and various metal sulphides in other submarine hydrothermal vent systems 70 , showing that BaSO 4 has the heaviest S-isotope composition. BaSO 4 is precipitated by the combination of Ba 2+ and SO 4 2− in hydrothermal vents, and its S-isotope composition is similar to that of SO 4 2− in the vent environment, indicating that SO 4 2− has a relatively heavy S-isotope composition in vent systems. In addition, the strong negative anomaly of δ 13 C -PDB reflected the selective absorption of light C isotopes by SRB.

Precipitation of metallic minerals in hydrothermal vents. Submarine hydrothermal vent fluids are
often rich in Fe 2+ , Pb 2+ , Zn 2+ , and other metal cations 2,71 . With drastic changes in the external physical and chemical environment, metal cations in hydrothermal fluids can easily combine with S, resulting in metal sulphide precipitation and accumulation 2 . Hydrogen sulphide (H 2 S) is an important factor that induces Fe ion precipitation; Fe ions can react with H 2 S to form FeS, as shown in Eq. (1) 13 : H 2 S can be formed in submarine hydrothermal vents by both inorganic and organic processes. SO 4 2− pyrolysis in vent hydrothermal fluids can release H 2 S, which exhausts the vents 63,64 . The organic production of H 2 S in submarine hydrothermal vents is related to SRB, the primary producers of the vent ecosystem, which are both chemoautotrophic and organo-heterotrophic. H 2 or organic matter can be used as an electron donor and SO 4 2− as (1) Fe 2+ + H 2 S = FeS + 2H + www.nature.com/scientificreports/ an electron acceptor to reduce SO 4 2− to H 2 S and obtain energy in the reaction process. These reactions are given in Eqs. (2) and (3), respectively 72 : H 2 S generated by organic and inorganic processes near hydrothermal vents rapidly combines with metal cations to form metal sulphides. Fe is first precipitated in the form of unstable mackinawite, greigite, or FeS; Zn and Pb form sphalerite (ZnFeS 2 ), galena (PbS 2 ), and other metal sulphides. The HCO 3 − generated by the reaction in Eq. (3) easily combines with Ca 2+ and/or Mg 2+ ions in the ocean and is deposited in the form of CaCO 3 or dolomite.
At present, two main FeS 2 formation pathways are known: polysulfide (S n 2− ) Eq. (4) and H 2 S Eq. (5) 60,73 : S n 2− and H 2 S can be produced in marine environments by inorganic processes or by processes involving microbes. In the S n 2− pathway, FeS aq is attacked by nucleophilic polysulphides to form FeS 2 (Eq. 4). In the H 2 S pathway, FeS 2 is formed by electron transfer via the inner sphere complex between FeS and H 2 S (Eq. 5) 60,73 . The O 2 -deficient and sulphur-rich environment of the early Earth can satisfy both reactions, and the conversion of FeS to FeS 2 is considered to be the key energy transfer reaction for the emergence of life 74,75 . SRB can efficiently synthesise S n 2− and H 2 S and promote FeS 2 formation. We infer that the precipitation of FeS is caused by Fe 2+ and H 2 S reactions (a mixture of H 2 S generated by inorganic and organic processes) in hydrothermal vents, whereas the formation of tubular and spherical FeS 2 is related to the presence and metabolic activities of SRB in the two massive sulphide ores within the Tongken Sn polymetallic deposit.

SRB growth pattern. Microscopic observations show that SRB extended and expanded in peripheral FeS
in the form of filaments, tubes, and spheroids (Fig. 2). As this is difficult to achieve in consolidated FeS, the most likely explanation is that precipitation of FeS and the activities of SRB took place concurrently. Specifically, as Fe 2+ and H 2 S react to form FeS precipitates in hydrothermal vents, SRB migrate from the micro-hydrothermal vent to colonise the surrounding area and synthesise FeS 2 . Subsequently, both FeS and FeS 2 precipitate with the co-occurrence of small amounts of SRB.
The EDX elemental maps revealed that the contents of C, N, and O in FeS were extremely low (Fig. 3f-h). Furthermore, in FeS, no distinct peak was observed within the 1000-1700 cm −1 range, which is where organic matter peaks typically manifest in the laser Raman spectrum ( Fig. 5a and b). Consequently, organic matter was nearly absent in FeS, suggesting minimal external organic matter input into the studied micro-hydrothermal vents. Therefore, the observed SRB may represent chemoautotrophic bacteria 76,77 , which rely on H 2 supplied by the vent hydrothermal fluid as an electron donor and on SO 4 2− as an electron acceptor to synthesise adenosine 5′-triphosphate in the cell to store energy needed for life, release H 2 S, and synthesise FeS 2 . Similar to the role of atmospheric CO 2 in photosynthesis, CO 2 derived from hydrothermal vent fluids can serve as the exclusive carbon source for cell synthesis during the growth of SRB 78 . The necessary N for SRB may come from N-containing ions such as NH 4 + , NO 3 − , or NO 2 − in the vent hydrothermal fluids. These ions can be obtained via processes such as nitrification, dissimilatory NO 3 − oxidation, or NO 2 − reduction [79][80][81] . Based on the morphological data of SRB traces obtained using polarised light microscopy and SEM, combined with the EDX analysis, isotopic analysis, and Raman spectroscopy, we inferred the SRB-mediated FeS 2 formation process in FeS under the theoretic framework of hydrothermal vent mineralisation. Microbes preserved in FeS 2 are chemoautotrophic SRB growing in hydrothermal vents on the seabed, and hydrothermal fluids provide the materials and energy needed for their growth. Such bacteria live near micro-hydrothermal vents and rely on H 2 as an electron donor and SO 4 2− as an electron acceptor in the hydrothermal fluid to produce energy and H 2 S. H 2 S combines with unconsolidated FeS around SRB to form FeS 2 . After ore consolidation, these tubular, beaded, and spherical FeS 2 structures are preserved within FeS, thus recording the morphological characteristics of SRB activity. SRB grow from micro-hydrothermal vents to their peripheries; tubular bodies may branch and thicken, sometimes spheroids may develop, and some spheroids may grow and proliferate directly from the micro-vents (Fig. 6). Tubular bodies are often interconnected between adjacent micro-vents (Fig. 2a-e). FeS 2 synthesised by SRB via sulphate reduction presents the above microbial trace characteristics enclosed within the FeS matrix. Although H 2 S generated by microbial metabolic processes may promote the precipitation and mineralisation of galena, sphalerite, chalcopyrite, and other metal sulphides, no biomorphs of SRB have been found in these metal sulphides; this is worth further exploration in future studies.

Conclusions
The genesis of the Tongkeng Sn polymetallic deposit is related to the mineralisation of Devonian submarine hydrothermal vent sediments. We investigated ores that contained both morphological and chemical evidence of SRB. Tubular, beaded, and spherical FeS 2 structures were discovered in close proximity to micro-hydrothermal vents, displaying morphological characteristics typical of SRB flora. By analysing the elemental composition, employing Raman spectroscopy, and conducting isotopic analysis, we confirmed that the formation of FeS 2 is a result of the metabolic activities of SRB, which involves reduction of SO 4 2− and production of H 2 S. We also (2)

Materials and methods
Sample collection. We performed a systematic geological survey and sampling work in the Tongkeng Sn polymetallic deposit. A total of 70 massive sulphide ore samples were collected and transformed into polished thin sections for microscopic identification and chemical composition analysis. Using a polarising microscope, we found SRB trace fossils in two samples. These two samples were numbered TK-51 and TK-53 and were taken from the No. 92 ore body within the ore-bearing Liujiang Formation (D3L). Raman microspectroscopy. Micro-Raman spectra were collected using an inVia Qontor confocal Raman instrument (Renishaw Plc, UK). The laser beam was focused on the sample through a 50× objective lens with 532 nm radiation provided by a solid-state laser. The laser power was set at 10 mW on the sample surface. Baseline correction and spectral peak fitting of the Raman spectra of all specimens were performed using Origin Pro-2021 (Learning Edition) and PeakFit (v. 4.12).

SEM-EDX.
Isotope ratio mass spectroscopy. Three sub-samples for stable isotope analysis were obtained from sample TK-53; phases analysed included FeS, FeS 2 , CaCO 3 , and organic C, and the elements analysed were Fe, S, C, and O. The three ore samples were crushed and sieved until the particle diameters were < 0.425 mm; FeS was first adsorbed with a magnet and then screened with a binocular microscope (20× magnification) to obtain FeS without impurities. FeS 2 , CaCO 3 , and non-metallic particles with high carbonaceous content were selected from non-magnetic mineral particles by screening using a binocular microscope. The three groups of FeS and FeS 2 samples were powdered for Fe and S stable isotopic analysis. Fe stable isotope analysis of FeS and FeS 2 was performed by Guangzhou ALS Chemex (China). S stable isotope analyses were performed in the stable isotope analysis laboratory of the Kunming University of Science and Technology (China). S in FeS and FeS 2 was converted to SO 2 by a high-temperature combustion method using an elemental analyser (Vario isotope cube, Elementar, Germany), which was then transferred into a gas-phase isotope ratio mass spectrometer (Isoprime-100, Elementar) to analyse its S-isotope composition.
The C and O isotopic components of CaCO 3 were converted to CO 2 by the phosphoric acid method using Isoprime multiflow equipment in the stable isotope analysis laboratory of Kunming University of Science and Technology, which was then transferred into a gas-phase isotope ratio mass spectrometer (Isoprime-100) to analyse its C and O stable isotope composition.
Non-metallic particles with a high C content were soaked in dilute hydrochloric acid, washed, and dried twice to remove carbonate minerals. In the stable isotope analysis laboratory of the Kunming University of Science and Technology, the organic C present in ores was converted into CO 2 using a high-temperature combustion method, which was then transferred into a gas isotope ratio mass spectrometer (Isoprime-100) to analyse its C stable isotope composition. Figure 6. Sulphate-reducing bacteria growth modes. Sulphate-reducing bacteria (green dots) growing at a micro-hydrothermal vent (grey); chemoautotrophic SRB proliferate, branch, and expand around the microhydrothermal vent in tubular and spherical shapes. Yellow represents pyrite (FeS 2 ), orange represents pyrrhotite (FeS), and blue represents seawater.

Data availability
All data that support the findings of this study are available in the paper. Source data are provided with this paper.